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. 2024 Dec;46(6):1303-1318.
doi: 10.1007/s10529-024-03517-9. Epub 2024 Jul 27.

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants

Flavia Fagiani #  1 , Rachele Frigerio #  1 , Anna Maria Salzano  2 , Andrea Scaloni  2 , Carla Marusic  3 , Marcello Donini  4
Affiliations

Plant production of recombinant antigens containing the receptor binding domain (RBD) of two SARS-CoV-2 variants

Flavia Fagiani et al. Biotechnol Lett. 2024 Dec.

Abstract

Objectives: The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc).

Results: The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35 mg/kg leaf fresh weight).

Conclusions: Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics.

Keywords: COVID19; Molecular farming; Proteolytic degradation; SARS-CoV-2; Transient expression.

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